1. Field of Invention
This invention relates generally to internalization of membrane proteins and in particular to internalization mediated by a methionine/leucine internalization motif.
2. Related Art
The endocytic pathway includes internalization of the receptor-ligand complex via clathrin-coated pits and accumulation in the endosomes. The receptor-ligand complex then dissociates in the endosomes and the dissociated molecules are either recycled back to the cell surface or targeted to lysosomes for degradation. Targeting of most receptors to coated pits and their traffic through endocytic compartments are generally mediated by endocytic signals located in the cytoplasmic domain of proteins. These signals fall into two major categories, tyrosine-based and di-leucine based signals.
The tyrosine-based signals are represented by NPXY and YXXΦ consensus motifs with the Y residue being critical for their function. NPXY signals mediate internalization of several type-1 membrane proteins such as LDL receptor, epidermal growth factor receptor, insulin receptor and others. The YXXΦ (Φ-bulky hydrophobic side chain) signals mediate internalization and lysosomal targeting of several type I and type II membrane proteins such as transferrin receptor, mannose-6-phosphate receptor, asialoglycoprotein receptor, polymeric immunoglobulin receptor and others.
The di-leucine based signals require two consecutive leucines or a leucine-isoleucine pair for their function. Studies have identified two distinct classes of di-leucine based signals represented by [DE]XXXL[LI] and DXXLL consensus sequences. Both of these signals are involved in internalization and lysosomal targeting of several membrane proteins. Proteins such as CD3-γ, LIMP-II, tyrosinase CD4, GLUT4 have a [DE]XXXL type signal whereas a DXXLL signal has been characterized in mannose 6-phosphate/insulin-like growth factor-II receptor, the cation-dependent mannose-6-phosphate receptor, LDL-receptor related proteins, β-secretase and others.
The membrane protein Prostate Specific Membrane Antigen (PSMA) was originally identified by the monoclonal antibody 7E11-C5 raised against the human prostate cancer cell line LNCaP. Subsequently, the PSMA gene was cloned (Israeli et al., 1993), and mapped to chromosome 11q. PSMA is a type II membrane protein with a short cytoplasmic N-terminal region (19 amino acids), a transmembrane domain (24 amino acids) and a large extracellular C-terminal portion (707 amino acids) with several potential N-glycosylation sites. It has been shown that PSMA is homologous to glutamate carboxypeptidase II (85% at nucleic acid level) isolated from rat brain, has folate hydrolase activity and N-acetylated α-linked acidic dipeptidase (NAALDase) activity. The extracellular domain of PSMA shows homology (26% identity at the amino acid level) to the transferrin receptor I and to a cloned transferrin receptor II.
PSMA has been the subject of interest in cancer research due to its potential as a diagnostic and therapeutic target for human prostate cancer. PSMA is abundantly expressed in prostate cancer cells. Its expression is further increased in higher-grade cancers, metastatic disease, and hormone-refractory prostate carcinoma. In addition, PSMA has become the focus of even more intense interest due to the findings that it is selectively expressed in the neovasculature of nearly all types of solid tumors, but not in the vasculature of normal tissue. The function of PSMA with respect to vascular endothelial cell biology and the direct correlation between its expression and increasing tumor aggressiveness in prostate cancer are intriguing. The use of antibodies against PSMA for immunotherapy of prostate cancer is the subject of research investigation.
The development of new therapeutic agents for the treatment of cancer and other diseases is often limited by an inability of the agents to cross the cell membrane. Size, charge and chemical composition are some of the factors responsible for this inability. Although methods such as liposome delivery and viral delivery, including adenovirus or lentivirus mediated delivery, can potentially provide therapeutic agents to cells, there is a continuing need to develop additional ways to deliver therapeutic agents.